Zinc finger proteins for DNA binding and gene regulation in plants

ABSTRACT

Disclosed herein are modified plant zinc finger proteins; compositions comprising modified plant zinc finger proteins and methods of making and using modified plant zinc finger proteins. The modified plant zinc finger proteins, in contrast to naturally-occurring plant zinc finger proteins, have a binding specificity that is determined by tandem arrays of modular zinc finger binding unit.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/055,713 filed Jan. 22, 2002, now U.S. Pat. No. 7,273,923 which claimsthe benefit of U.S. provisional patent application Ser. No. 60/263,445filed Jan. 22, 2001 and U.S. provisional patent application Ser. No.60/290,716 filed May 11, 2001. The disclosures of the aforementionedapplications are hereby incorporated by reference in their entiretiesfor all purposes.

TECHNICAL FIELD

The methods and compositions disclosed herein relate generally to thefield of regulation of gene expression and specifically to methods ofmodulating gene expression in plants by utilizing polypeptides derivedfrom plant zinc finger-nucleotide binding proteins.

BACKGROUND

Zinc finger proteins (ZFPs) are proteins that bind to DNA, RNA and/orprotein, in a sequence-specific manner, by virtue of a metal stabilizeddomain known as a zinc finger. See, for example, Miller et al. (1985)EMBO J. 4:1609-1614; Rhodes et al. (1993) Sci. Amer. February:56-65; andKlug (1999) J. Mol. Biol. 293:215-218. There are at least 2 classes ofZFPs which co-ordinate zinc to form a compact DNA-binding domain. Eachclass can be distinguished by the identities of the conservedmetal-binding amino acids and by the associated architecture of theDNA-binding domain.

The most widely represented class of ZFPs, known as the C₂H₂ ZFPs,comprises proteins that are composed of zinc fingers that contain twoconserved cysteine residues and two conserved histidine residues. Over10,000 C₂H₂ zinc fingers have been identified in several thousand knownor putative transcription factors. Each C₂H₂ zinc finger domaincomprises a conserved sequence of approximately 30 amino acids thatcontains the invariant cysteines and histidines in the followingarrangement: -Cys-(X)₂₋₄-Cys-(X)₁₂-His-(X)₃₋₅-His (SEQ ID NO: 1). Inanimal genomes, polynucleotide sequences encoding this conserved aminoacid sequence motif are usually found as a series of tandemduplications, leading to the formation of multi-finger domains within aparticular transcription factor.

Several structural studies have demonstrated that the conserved C₂H₂amino acid motif folds into a beta turn (containing the two invariantcysteine residues) and an alpha helix (containing the two invarianthistidine residues). The alpha helix and beta turn associate along ahydrophobic interface and are held together through the tetrahedralcoordination of a zinc atom by the conserved cysteines and histidines.

The three-dimensional structure of a complex between a DNA target siteand a polypeptide comprising three C₂H₂ zinc fingers derived from themouse immediate early protein zif268 (also known as Krox-24) has beendetermined by x-ray crystallography. Pavletich et al. (1991) Science252:809-817. The structure reveals that the amino acid side chains oneach zinc finger alpha helix interact specifically with functionalgroups of the nucleotide bases exposed in the DNA major groove. Eachfinger interacts with DNA as a module; changes in the sequence of aminoacids of the recognition helix can result in corresponding changes intarget site specificity. See, for example, Wolfe et al. (1999) Annu.Rev. Biophys. Biomol. Struct. 3:183-212.

Another class of ZFPs includes the so-called C₃H ZFPs. See, e.g., Jianget al. (1996) J. Biol. Chem. 271:10723-10730 for a discussion ofCys-Cys-His-Cys (C₃H) ZFPs.

The modular nature of sequence-specific interactions between zincfingers and DNA sequences (i.e., a particular zinc finger of definedsequence binds to a DNA triplet or quadruplet of defined sequence)allows certain DNA-binding domains of predetermined specificity to bedesigned and/or selected. See, for example, Blackburn (2000) Curr. Opin.Struct. Biol. 10:399-400; Segal et al. (2000) Curr. Opin. Chem. Biol.4:34-39. To this end, numerous modifications of animal C₂H₂ zinc fingerproteins, most often either mouse zif268 or human SP-1, have beenreported. See, e.g., U.S. Pat. Nos. 6,007,988; 6,013,453; 6,140,081;6,140,466; GB Patent No. 2,348,424; PCT WO98/53057; PCT WO98/53058; PCTWO98/53059; PCT WO98/53060; PCT WO98/54311; PCT WO00/23464; PCT WO00/42219; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; Segalet al. (2000) supra; and references cited in these publications. Theresults of these and other studies are generally consistent with theidea that it is possible to obtain C₂H₂ ZFPs, based on, for example, themouse zif268 ZFP or the human SP-1 ZFP, of desired target sitespecificity. Such target-specific ZFPs are generally obtained byselection or design of individual fingers, each of which has a 3-4nucleotide target specificity, and assembly of such fingers into a ZFPhaving a target site specificity of 9-20 nucleotides.

C₂H₂ ZFPs have been identified in plants, where they are involved in,for example, developmental regulation of various floral and vegetativeorgans. See, e.g., Takatsuji (1999) Plant Mol. Biol. 39:1073-1078. Inplant ZFPs, however, zinc fingers do not generally occur inclosely-spaced tandem arrays. For example, in a family of DNA bindingproteins identified in Petunia (the EPF family), two canonical Cys₂-His₂zinc finger motifs are separated by an intervening stretch of between 19and 232 amino acids. The binding capability of this class of proteinsappears to be determined by both the zinc fingers and the interveningamino acids, suggesting that plant zinc finger proteins have a differentmechanism of DNA binding that do the zif268 and SP-1 zinc fingerproteins, for example. In addition, the sequence specificity of DNAbinding by EPF-type plant ZFPs is dependent upon different positions inthe recognition helix of the zinc finger than is the specificity of DNAbinding by most zif268-type ZFPs. See, for example, Takatsuji (1996)Biochem. Biophys. Res. Comm. 224:219-223.

Targeted gene regulation in plants would facilitate numerousapplications such as, for example, the optimization of crop traitsaffecting nutritional value, yield, stress tolerance, pathogenresistance, and resistance to agrochemicals. In addition, targeted generegulation could be used to study gene function in plants, and to adaptplants for use as biological factories for the production ofpharmaceutical compounds or industrial chemicals. Such regulation couldtheoretically be achieved by design of plant transcriptional regulatoryproteins of predetermined DNA sequence specificity. However, to date,naturally occurring plant ZFPs that recognize DNA by using a tandemarrangement of modular zinc finger binding domains (as do zif268 andrelated ZFPs) have not been described. Therefore, it remains difficult,if not impossible, to design a plant ZFP capable of recognizing andbinding to a particular predetermined nucleotide sequence. Furthermore,since the mechanism of DNA binding by plant ZFPs remains largelyunknown, no immediate solution to this problem is apparent. Accordingly,the ability to design and/or select plant zinc finger proteins ofpredetermined target specificity would be desirable.

SUMMARY

The present disclosure provides plant DNA-binding proteins that aremodified in such a way that their mechanism and specificity of DNAbinding are determined by tandem arrays of modular zinc finger bindingunits. In this way, design strategies and selection methods which havebeen developed and utilized for other classes of ZFPs can be applied tothe production of plant ZFPs having a predetermined target sitespecificity, for use in modulation of gene expression in plant cells.

In one aspect, disclosed herein is a modified plant zinc finger protein(ZFP) that binds to a target sequence. The target sequence can be, forexample, nucleic acid (DNA or RNA) or amino acids of any length, forinstance 3 or more contiguous nucleotides. In certain embodiments, themodified plant ZFP comprises a tandem array of zinc fingers. One, morethan one or all of the zinc fingers of the ZFP may be naturallyoccurring or may be obtained by rational design and/or selection (e.g.,phage display, interaction trap, ribosome display and RNA-peptidefusion. Thus, in certain embodiments, one or more of the zinc fingerscomprise canonical C₂H₂ zinc fingers and in other embodiments, one ormore of the zinc fingers comprise non-canonical zinc fingers. In any ofthe modified plant ZFPs described herein, one or more of the zincfingers are derived from two or more plant species, for example, bydeleting and/or substituting one or more amino acid residues as comparedto a naturally occurring plant ZFP. In certain embodiments, one or moreamino acid residues are deleted between one or more of the zinc fingers.

Thus, in one embodiment, plant zinc finger proteins (ZFPs) are modified,for example, by deletion of inter-zinc finger sequences and/or insertionof additional zinc finger sequences, to generate one or more tandemarrays of zinc fingers. Thus, in contrast to naturally occurring plantzinc finger proteins, their mechanism and specificity of DNA binding aredetermined by tandem arrays of modular zinc finger binding units. Inanother embodiment, plant zinc fingers of disparate origin (e.g., zincfingers from Petunia and Arabidopsis) are recombined into a tandem arrayof modular zinc finger binding units.

In yet another aspect, a fusion polypeptide comprising (i) a modifiedplant ZFP as described herein and (ii) at least one functional domainare described. The functional domain may be a repressive domain or anactivation domain.

In yet another aspect, isolated polynucleotides encoding any of themodified plant zinc finger proteins or fusion polypeptides describedherein are provided. Also provided are expression vectors comprisingthese polynucleotides. Also described are host cell comprising thesepolynucleotides and/or expression vectors.

In another aspect, a method for modulating gene expression in a plantcell comprising contacting the cell with any of the modified plant ZFPsdescribed herein is provided. In one embodiment, the protein comprisinga tandem array of zinc fingers is provided. Preferably, the protein isexpressed in the cell, for example, by introducing the protein and/or anucleic acid encoding the protein into the cell. In certain embodiments,the zinc fingers of the protein comprise an adapted amino acid sequenceat any one or more of residues −1 through +6 of the recognition helix.The adapted amino acid sequence can be obtained by rational designand/or by selection (e.g., using methods such as phage display,interaction trap, ribosome display, RNA-peptide fusion or combinationsof one or more of these methods). In certain embodiments, the proteincomprises zinc finger backbones from different species, for exampledifferent plant species. In other embodiments, the protein compriseszinc finger backbones of plant origin, fungal origin or combinationsthereof.

Furthermore, in certain embodiments, the protein is obtained by deletionof inter-finger sequences from a plant ZFP.

In other aspects, the methods described herein make use of a fusionprotein comprising a tandem array of zinc fingers and one or morefunctional domains, for example, one or more transcriptional activation(e.g., C1, etc.) or repression domains.

In other aspects, the compositions and methods described herein find usein a variety of applications in which modulation of gene expressionalters the phenotype and/or composition of the plant or plant cell, forexample by optimizing crop traits such as nutritional value, yield,stress tolerance, pathogen resistance, resistance to agrochemicals(e.g., insecticides and/or herbicides) and the like; and by adaptingplants for use in production of pharmaceutical compounds and/orindustrial chemicals. In certain embodiments, the modulation of geneexpression can be used to study genetic pathways and/or gene functionsin plants.

These and other embodiments will readily occur to those of skill in theart in light of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting construction of the YCF3 expressionvector useful in expressing modified plant ZFPs.

FIG. 2 shows the results of analysis of GMT mRNA in RNA isolated fromArabidopsis thaliana protoplasts that had been transfected withconstructs encoding fusion of a transcriptional activation domain withvarious modified plant ZFPs. Results are expressed as GMT mRNAnormalized to 18S rRNA. AGMT numbers on the abscissa refer to themodified plant ZFP binding domains shown in Table 2. Duplicate TaqMan®analyses are shown for each RNA sample.

DETAILED DESCRIPTION

General

The present disclosure provides modified plant ZFPs (and functionalfragments thereof), wherein zinc fingers are arranged in one or moretandem arrays such that, upon DNA binding, each zinc finger contacts atriplet or quadruplet target subsite. In preferred embodiments, thetarget subsites are contiguous to one another. The modified plant ZFPcan be a fusion polypeptide and, either by itself or as part of such afusion, can enhance or suppress expression of a gene (i.e., modulategene expression). Polynucleotides encoding modified plant ZFPs, andpolynucleotides encoding fusion proteins comprising one or more modifiedplant ZFPs are also provided. Additionally provided are compositionscomprising, in combination with an acceptable carrier, any of themodified plant zinc finger binding polypeptides described herein orfunctional fragments thereof; and compositions comprising a nucleotidesequence that encodes a modified plant zinc finger binding polypeptideor functional fragment thereof, wherein the modified plant zinc fingerpolypeptide or functional fragment thereof binds to a cellularnucleotide sequence to modulate the function of the cellular nucleotidesequence.

Currently, ZFPs targeted to specific predetermined sequences are derivedfrom non-plant ZFPs such as Xenopus TFIIIA, murine zif268, human SP-1and the like. Accordingly, in one embodiment, modified plant zinc fingerproteins, targeted to predetermined sequences, are described wherein allor substantially all of the sequences making up the ZFP are derived fromone or more plant sources. Furthermore, the modified plant ZFPs areorganized in non-plant ZFP structures, for example structures in whichindividual zinc fingers (e.g., C₂H₂ fingers) are linked by short linkersequences, or structures that do not contain native plant DNA bindingsequences such as inter-zinc finger sequences of a plant zinc fingerprotein, (which can be generated from plant ZFPs, for example, bydeletion of inter-zinc finger amino acid sequences). In certainembodiments, all amino acid residues of a modified plant ZFP are derivedfrom a non-modified plant ZFP (e.g., when a modified plant ZFP isobtained by deletion of inter-finger sequences from a non-modified plantZFP). In other embodiments, one or more amino acid residues of amodified plant ZFP (e.g., amino acids involved in sequence-specificand/or non-specific DNA contacts) can be either designed or selected,and thus may not constitute part of the original plant ZFP sequence.

It is preferred that a modified plant zinc finger protein be amulti-finger protein, for example comprising at least threezinc-coordinating fingers. In the standard nomenclature for ZFPs, the“first” finger is the N-terminal-most finger of the protein (withrespect to the other fingers) and binds to the 3′-most triplet (orquadruplet) subsite in the target site. Additional fingers, movingtowards the C-terminus of the protein, are numbered sequentially.

In other embodiments, one or more of the component fingers of themodified plant ZFP will be a non-C₂H₂ structure. For example, in certainembodiments, a three-finger zinc finger protein is provided wherein thefirst two fingers are of the C₂H₂ class but the third finger is non-C₂H₂(e.g., C₃H or other structure) as described, for example, inInternational Publication WO 02/57293.

Therefore, the modified plant ZFPs disclosed herein differ frompreviously described designed zinc finger protein transcription factorsin that they are entirely or primarily composed of plant sequences.Nonetheless, the plant sequences are assembled such that the overallstructure of the binding region of the modified plant protein is similarto that of a non-plant eukaryotic zinc finger. Thus, modified plantZFPs, as disclosed herein, comprise plant sequences either for theentire ZFP or for most of the ZFP. In the latter case, plant sequencesare used preferably in all regions except those residues involved inrecognition and/or binding to the target site, which can comprise, forexample, sequences obtained by rational design and/or selection.

It will be readily apparent that various combinations of zinc fingerscan be used in a single modified plant ZFP. For example, all of thefinger components can be designed (i.e., their sequences are obtained asa result of rational design methods); all of the finger components canbe selected (i.e., their sequences are obtained by a selection methodsuch as, e.g. phage display, two-hybrid systems or interaction trapassays); all of the finger components can be naturally-occurring plantzinc fingers; or the component fingers of a modified plant ZFP can beany combination of naturally-occurring plant zinc fingers, designedfingers and selected fingers.

In additional embodiments, the modified plant zinc finger proteinsdescribed herein (and/or functional fragments thereof) are used infusion proteins, for example fusions of a modified plant ZFP DNA-bindingdomain with, e.g., a repression domain, an activation domain, achromatin remodeling domain, a component of a chromatin remodelingcomplex, a methyl-binding domain, a methyltransferase, aninsulator-binding protein, and/or functional fragments thereof.Polynucleotides encoding any of the zinc finger proteins, componentsthereof, functional fragments thereof, and fusions thereof are alsoprovided.

In additional embodiments, methods for modulating gene expression inplant cells, using modified plant ZFPs are provided. Becausenaturally-occurring plant ZFPs, which modulate plant gene expression invivo, do not contain zinc fingers in tandem arrays, the ability of a ZFPcontaining a tandem array of zinc fingers to modulate gene expression ina plant cell is a surprising discovery. Thus, the compositions andmethods disclosed herein allow the insights gained from work withnon-plant ZFPs such as zif268 and Sp-1 to be applied to regulation ofplant gene expression by plant proteins; so that targeted regulation ofgene expression in plant cells can be achieved by mechanisms similar tothose already described for animal cells. In addition, by allowingtargeted regulation of plant gene expression by plant proteins, thepresent methods and compositions will help to allay potential concernsregarding the introduction of animal proteins into plants.

The practice of the disclosed methods employs, unless otherwiseindicated, conventional techniques in molecular biology, biochemistry,genetics, computational chemistry, cell culture, recombinant DNA andrelated fields as are within the skill of the art. These techniques arefully explained in the literature. See, for example, Sambrook et al.MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold SpringHarbor Laboratory Press, 1989; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodicupdates; and the series METHODS IN ENZYMOLOGY, Academic Press, SanDiego.

The disclosures of all patents, patent applications and publicationsmentioned herein are hereby incorporated by reference in theirentireties.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form. Forthe purposes of the present disclosure, these terms are not to beconstrued as limiting with respect to the length of a polymer. The termscan encompass known analogues of natural nucleotides, as well asnucleotides that are modified in the base, sugar and/or phosphatemoieties. In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a corresponding naturallyoccurring amino acid, for example selenocysteine (Bock et al. (1991)Trends Biochem. Sci. 16:463-467; Nasim et al. (2000) J. Biol. Chem.275:14,846-14, 852) and the like.

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity. A“binding profile” refers to a plurality of target sequences that arerecognized and bound by a particular binding protein. For example, abinding profile can be determined by contacting a binding protein with apopulation of randomized target sequences to identify a sub-populationof target sequences bound by that particular binding protein.

A “zinc finger binding protein” is a protein or segment within a largerprotein that binds DNA, RNA and/or protein in a sequence-specific manneras a result of stabilization of protein structure through coordinationof a zinc ion. The term zinc finger binding protein is often abbreviatedas zinc finger protein or ZFP.

A zinc finger “backbone” is the portion of a zinc finger outside theregion involved in DNA major groove interactions; i.e., the regions ofthe zinc finger outside of residues −1 through +6 of the alpha helix.The backbone comprises the beta strands, the connecting region betweenthe second beta strand and the alpha helix, the portion of the alphahelix distal to the first conserved histidine residue, and theinter-finger linker sequence(s). Thus, a plant zinc finger “backbone”refers to sequences derived from one or more plant ZFPs, where thesesequences are not naturally involved in DNA major groove interactions.

As used herein, the term “modified plant” zinc finger protein refers toa zinc finger protein comprising plant ZFP sequences organized in anon-plant ZFP structure, for example to eliminate the long stretches ofamino acid sequence between zinc fingers found in manynaturally-occurring plant ZFPs. Thus, all, most or some of the sequencesin the zinc finger regions of a modified plant ZFP may be derived from aplant. Additionally, modified plant ZFPs in these non-plant structurescan further include one or more residues or regions (e.g., fingers) ofnon-plant origin, such as, for example, naturally-occurring fingers orfingers as may be obtained by design or selection, so long as DNAbinding capability is maintained.

A “non-canonical” zinc finger protein is a protein not occurring innature that has been designed and/or selected so as to differ from thecanonical binding domain consensus sequence Cys-Cys-His-His (e.g.,Cys2-His2). Thus, non-canonical zinc finger proteins comprise asubstitution, addition and/or deletion of at least one amino acid,compared to a naturally occurring zinc finger protein. Non-limitingexamples of non-canonical zinc fingers include binding domainscomprising Cys-Cys-His-Cys (e.g., C3H) sequences and the like. (See,also International Publication WO 02/57293).

A “designed” zinc finger protein is a protein not occurring in naturewhose structure and composition results principally from rationalcriteria. Criteria for rational design include application ofsubstitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP designsand binding data, for example as described in co-owned PCT WO 00/42219.A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, two-hybrid systems and/or interaction trap assays. See e.g.,U.S. Pat. Nos. 5,789,538; 6,007,988; 6,013,453; WO 95/19431; WO96/06166; WO 98/54311 and Joung et al. (2000) Proc. Natl. Acad. Sci. USA97:7382-7387. Selection methods also include ribosome display systems(e.g., PCT WO 00/27878) and mRNA-peptide fusion systems (e.g., U.S. Pat.No. 6,207,446; PCT WO 00/47775). Amino acid sequences of polypeptides(e.g., zinc fingers) obtained by selection or design are referred to as“adapted” amino acid sequences. Designed and/or selected ZFPs aremodified according to the methods and compositions disclosed herein andmay also be referred to as “engineered” ZFPs.

The term “naturally-occurring” is used to describe an object that can befound in nature, as distinct from being artificially produced by ahuman. For example, naturally occurring plant ZFPs are characterized bylong spacers of diverse lengths between adjacent zinc finger components.

Nucleic acid or amino acid sequences are “operably linked” (or“operatively linked”) when placed into a functional relationship withone another. For instance, a promoter or enhancer is operably linked toa coding sequence if it regulates, or contributes to the modulation of,the transcription of the coding sequence. Operably linked DNA sequencesare typically contiguous, and operably linked amino acid sequences aretypically contiguous and in the same reading frame. However, sinceenhancers generally function when separated from the promoter by up toseveral kilobases or more and intronic sequences may be of variablelengths, some polynucleotide elements may be operably linked but notcontiguous. Similarly, certain amino acid sequences that arenon-contiguous in a primary polypeptide sequence may nonetheless beoperably linked due to, for example folding of a polypeptide chain.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a modifiedplant ZFP DNA-binding domain is fused to a functional domain (orfunctional fragment thereof), the ZFP DNA-binding domain and thefunctional domain (or functional fragment thereof) are in operativelinkage if, in the fusion polypeptide, the modified plant ZFPDNA-binding domain portion is able to bind its target site and/or itsbinding site, while the functional domain (or functional fragmentthereof) is able to modulate (e.g., activate or repress) transcription.

“Specific binding” between, for example, a ZFP and a specific targetsite means a binding affinity of at least 1×10⁶ M⁻¹.

A “fusion molecule” is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion polypeptides (for example, a fusion between amodified plant ZFP DNA-binding domain and a functional domain) andfusion nucleic acids (for example, a nucleic acid encoding the fusionpolypeptide described herein). Examples of the second type of fusionmolecule include, but are not limited to, a fusion between atriplex-forming nucleic acid and a polypeptide, and a fusion between aminor groove binder and a nucleic acid.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see below), as well as all DNA regionsthat regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions. Further, apromoter can be a normal cellular promoter or, for example, a promoterof an infecting microorganism such as, for example, a bacterium or avirus.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Gene activation” and “augmentation of gene expression” refer to anyprocess that results in an increase in production of a gene product. Agene product can be either RNA (including, but not limited to, mRNA,rRNA, tRNA, and structural RNA) or protein. Accordingly, gene activationincludes those processes that increase transcription of a gene and/ortranslation of an mRNA. Examples of gene activation processes whichincrease transcription include, but are not limited to, those whichfacilitate formation of a transcription initiation complex, those whichincrease transcription initiation rate, those which increasetranscription elongation rate, those which increase processivity oftranscription and those which relieve transcriptional repression (by,for example, blocking the binding of a transcriptional repressor). Geneactivation can constitute, for example, inhibition of repression as wellas stimulation of expression above an existing level. Examples of geneactivation processes that increase translation include those thatincrease translational initiation, those that increase translationalelongation and those that increase mRNA stability. In general, geneactivation comprises any detectable increase in the production of a geneproduct, preferably an increase in production of a gene product by about2-fold, more preferably from about 2- to about 5-fold or any integralvalue therebetween, more preferably between about 5- and about 10-foldor any integral value therebetween, more preferably between about 10-and about 20-fold or any integral value therebetween, still morepreferably between about 20- and about 50-fold or any integral valuetherebetween, more preferably between about 50- and about 100-fold orany integral value therebetween, more preferably 100-fold or more.

“Gene repression” and “inhibition of gene expression” refer to anyprocess that results in a decrease in production of a gene product. Agene product can be either RNA (including, but not limited to, mRNA,rRNA, tRNA, and structural RNA) or protein. Accordingly, gene repressionincludes those processes that decrease transcription of a gene and/ortranslation of an mRNA. Examples of gene repression processes whichdecrease transcription include, but are not limited to, those whichinhibit formation of a transcription initiation complex, those whichdecrease transcription initiation rate, those which decreasetranscription elongation rate, those which decrease processivity oftranscription and those which antagonize transcriptional activation (by,for example, blocking the binding of a transcriptional activator). Generepression can constitute, for example, prevention of activation as wellas inhibition of expression below an existing level. Examples of generepression processes that decrease translation include those thatdecrease translational initiation, those that decrease translationalelongation and those that decrease mRNA stability. Transcriptionalrepression includes both reversible and irreversible inactivation ofgene transcription. In general, gene repression comprises any detectabledecrease in the production of a gene product, preferably a decrease inproduction of a gene product by about 2-fold, more preferably from about2- to about 5-fold or any integral value therebetween, more preferablybetween about 5- and about 10-fold or any integral value therebetween,more preferably between about 10- and about 20-fold or any integralvalue therebetween, still more preferably between about 20- and about50-fold or any integral value therebetween, more preferably betweenabout 50- and about 100-fold or any integral value therebetween, morepreferably 100-fold or more. Most preferably, gene repression results incomplete inhibition of gene expression, such that no gene product isdetectable.

The term “modulate” refers to a change in the quantity, degree or extentof a function. For example, the modified plant zinc finger-nucleotidebinding polypeptides disclosed herein can modulate the activity of apromoter sequence by binding to a motif within the promoter, therebyinducing, enhancing or suppressing transcription of a gene operativelylinked to the promoter sequence. Alternatively, modulation may includeinhibition of transcription of a gene wherein the modified zincfinger-nucleotide binding polypeptide binds to the structural gene andblocks DNA dependent RNA polymerase from reading through the gene, thusinhibiting transcription of the gene. The structural gene may be anormal cellular gene or an oncogene, for example. Alternatively,modulation may include inhibition of translation of a transcript. Thus,“modulation” of gene expression includes both gene activation and generepression.

Modulation can be assayed by determining any parameter that isindirectly or directly affected by the expression of the target gene.Such parameters include, e.g., changes in RNA or protein levels; changesin protein activity; changes in product levels; changes in downstreamgene expression; changes in transcription or activity of reporter genessuch as, for example, luciferase, CAT, beta-galactosidase, or GFP (see,e.g., Mistili & Spector, (1997) Nature Biotechnology 15:961-964);changes in signal transduction; changes in phosphorylation anddephosphorylation; changes in receptor-ligand interactions; changes inconcentrations of second messengers such as, for example, cGMP, cAMP,IP₃, and Ca2⁺; changes in cell growth, changes in chemical composition(e.g., nutritional value), and/or changes in any functional effect ofgene expression. Measurements can be made in vitro, in vivo, and/or exvivo. Such functional effects can be measured by conventional methods,e.g., measurement of RNA or protein levels, measurement of RNAstability, and/or identification of downstream or reporter geneexpression. Readout can be by way of, for example, chemiluminescence,fluorescence, colorimetric reactions, antibody binding, induciblemarkers, ligand binding assays; changes in intracellular secondmessengers such as cGMP and inositol triphosphate (IP₃); changes inintracellular calcium levels; cytokine release, and the like.

“Eucaryotic cells” include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells.Similarly, “prokaryotic cells’ include, but are not limited to,bacteria.

A “regulatory domain” or “functional domain” refers to a protein or apolypeptide sequence that has transcriptional modulation activity, orthat is capable of interacting with proteins and/or protein domains thathave transcriptional modulation activity. Typically, a functional domainis covalently or non-covalently linked to a ZFP to modulatetranscription of a gene of interest. Alternatively, a ZFP can act, inthe absence of a functional domain, to modulate transcription.Furthermore, transcription of a gene of interest can be modulated by aZFP linked to multiple functional domains.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well known in the art. Similarly,methods for determining protein function are well known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. See Ausubel et al., supra. The ability of aprotein to interact with another protein can be determined, for example,by co-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

A “target site” or “target sequence” is a sequence that is bound by abinding protein such as, for example, a ZFP. Target sequences can benucleotide sequences (either DNA or RNA) or amino acid sequences. By wayof example, a DNA target sequence for a three-finger ZFP is generallyeither 9 or 10 nucleotides in length, depending upon the presence and/ornature of cross-strand interactions between the ZFP and the targetsequence. Target sequences can be found in any DNA or RNA sequence,including regulatory sequences, exons, introns, or any non-codingsequence.

A “target subsite” or “subsite” is the portion of a DNA target site thatis bound by a single zinc finger, excluding cross-strand interactions.Thus, in the absence of cross-strand interactions, a subsite isgenerally three nucleotides in length. In cases in which a cross-strandinteraction occurs (e.g., a “D-able subsite,” as described for examplein co-owned PCT WO 00/42219, incorporated by reference in its entiretyherein) a subsite is four nucleotides in length and overlaps withanother 3- or 4-nucleotide subsite.

The term “effective amount” includes that amount which results in thedesired result, for example, deactivation of a previously activatedgene, activation of a previously repressed gene, or inhibition oftranscription of a structural gene or translation of RNA.

Zinc Finger Proteins

Zinc finger proteins are polypeptides that comprise zinc fingercomponents. For example, zinc finger proteins can have one tothirty-seven fingers, commonly having 2, 3, 4, 5 or 6 fingers. Zincfinger DNA-binding proteins are described, for example, in Miller et al.(1985) EMBO J. 4:1609-1614; Rhodes et al. (1993) Scientific AmericanFebruary:56-65; and Klug (1999) J. Mol. Biol. 293:215-218. A zinc fingerprotein recognizes and binds to a target site (sometimes referred to asa target sequence or target segment) that represents a relatively smallportion of sequence within a target gene. Each component finger of azinc finger protein binds to a subsite within the target site. Thesubsite includes a triplet of three contiguous bases on the same strand(sometimes referred to as the target strand). The three bases in thesubsite can be individually denoted the 5′ base, the mid base, and the3′ base of the triplet, respectively. The subsite may or may not alsoinclude a fourth base on the non-target strand that is the complement ofthe base immediately 3′ of the three contiguous bases on the targetstrand. The base immediately 3′ of the three contiguous bases on thetarget strand is sometimes referred to as the 3′ of the 3′ base.Alternatively, the four bases of the target strand in a four basesubsite can be numbered 4, 3, 2, and 1, respectively, starting from the5′ base.

Zinc finger proteins have been identified in a variety of species. Whileplant ZFPs are characterized by long spacers between fingers, non-plantZFPs have much shorter linkers between-finger regions. An exemplarynon-plant ZFP is the human transcription factor, Sp-1. As described indetail in WO 00/42219, each of the three zinc fingers in Sp-1 isapproximately 30 amino acids in length and is made up of a beta turn(approximately 12 residues in length), and alpha helix (approximately10-12 residues in length) and short sequence connecting between the betaturn and the alpha helix of approximately 2 residues and an inter-fingerlinker sequence of 4-5 residues. Exemplary sequences of the zinc fingersof Sp-1 are shown in co-owned WO 00/42219. Also disclosed in WO 00/42219is an SP-1 consensus sequence, as described by Berg (1992) Proc. Natl.Acad. Sci. USA 89:11,109-11,110, which is useful in the design oftargeted zinc finger proteins.

Furthermore, in discussing the specificity-determining regions of a zincfinger, amino acid +1 refers to the first amino acid in thealpha-helical portion of the zinc finger. The portion of a zinc fingerthat is generally believed to be responsible for its binding specificitylies between −1 and +6. Amino acid ++2 refers to the amino acid atposition +2 in a second zinc finger adjacent (in the C-terminaldirection) to the zinc finger under consideration. In certaincircumstances, a zinc finger binds to its triplet subsite substantiallyindependently of other fingers in the same zinc finger protein.Accordingly, the binding specificity of a zinc finger protein containingmultiple fingers is, to a first approximation, the aggregate of thespecificities of its component fingers. For example, if a zinc fingerprotein is formed from first, second and third fingers that individuallybind to triplets XXX, YYY, and ZZZ, the binding specificity of the zincfinger protein is 3′-XXX YYY ZZZ-5′.

The relative order of fingers in a zinc finger protein, from N-terminalto C-terminal, determines the relative order of triplets in the targetsequence, in the 3′ to 5′ direction that will be recognized by thefingers. For example, if a zinc finger protein comprises, fromN-terminal to C-terminal, first, second and third fingers thatindividually bind to the triplets 5′-GAC-3′,5′-GTA-3′ and 5′-GGC-3′,respectively, then the zinc finger protein binds to the target sequence5′-GGCGTAGAC-3′ (SEQ ID NO: 2). If the zinc finger protein comprises thefingers in another order, for example, second finger, first finger,third finger, then the zinc finger protein binds to a target segmentcomprising a different permutation of triplets, in this example,5′-GGCGACGTA-3′ (SEQ ID NO: 3). See Berg et al. (1996) Science271:1081-1086. The numbering convention used above is standard in thefield for the region of a zinc finger conferring binding specificity.The amino acid on the N-terminal side of the first invariant His residueis assigned the number +6, and other amino acids, proceeding in anN-terminal direction, are assigned successively decreasing numbers. Thealpha helix generally begins at residue +1 and extends to the residuefollowing the second conserved histidine. The entire helix can thereforebe of variable length, e.g., between 11 and 13 residues.

A. Modified Plant ZFPs

A modified plant zinc finger protein is an amino acid sequence, orvariant or fragment thereof, which is capable of binding to a targetsequence and which comprises sequences derived from plant sources whichhave been reassembled in a non-plant ZFP structure. Thus, one or more ofthe following regions of a modified plant zinc finger are derived fromone or more plant sources: the first beta strand, the second betastrand, the alpha helix, and the linker.

It is to be understood that “non-plant” structure refers to anystructure that deviates from typical naturally occurring plant ZFPs. Oneexample of a non-plant ZFP scaffold suitable for providing a templatefor assembling plant-derived sequences is one in which the number ofresidues between the second histidine of one finger and the firstcysteine of the adjacent, C-terminal finger is relatively short. Incontrast to typical non-plant ZFPs, plant ZFPs are characterized by longspacers between adjacent fingers. Thus, in certain embodiments, anon-plant structure refers to ZFPs which contain tandem arrays of zincfingers, i.e., structures in which there are between 5 and 50 aminoacids between fingers, more preferably between 5 and 25 amino acids andeven more preferably between 5 and 20 amino acids, or any integertherebetween.

Thus, in certain embodiments, the modified plant ZFPs disclosed hereinwill not contain the sequence QALGGH (SEQ ID NO:105) in the recognitionregion, which is highly conserved in many plant ZFPs. See Takatsuji,(1999) Plant Mol. Biol. 39:1073-1078 and references cited therein. Yetanother example of a non-plant ZFP structure is one that comprises bothcanonical C₂H₂ fingers and non-canonical (e.g., non-C₂H₂) fingers. (See,also International Publication WO 02/57293). Other examples of non-plantstructures can be readily determined by those of skill in the art inview of the teachings herein. Furthermore, it is to be understood thatthe modified plant ZFPs described herein may have one or more of thesenon-plant organization characteristics.

Thus, although the modified plant ZFPs disclosed herein are composedwholly or partly of plant sequences, they have a non-plant structure.The non-plant structure of the modified plant ZFP can be similar to thatof any class of non-plant ZFP, for instance the C₂H₂ canonical class ofZFPs as exemplified by TFIIIA, Zif268 and Sp-1. Furthermore, themodified plant ZFP can comprise sequences from more than one class ofZFP, and selecting particular DNA binding residues and plant backboneresidues to achieve the desired effector functions is within theordinary skill in the art. The Sp-1 sequence used for construction oftargeted zinc finger proteins corresponds to amino acids 531 to 624 inthe Sp-1 transcription factor. Thus, models for design of modified plantZFPs include, but are not limited to, Sp-1 and an Sp-1 consensussequence, described by Berg (1992) Proc. Natl. Acad. Sci. USA89:11,109-11,110 and by Shi et al. (1995) Chemistry and Biology 1:83-89.The amino acid sequences of these ZFP frameworks are disclosed inco-owned PCT WO 00/42219, the disclosure of which is incorporated byreference. Fungal ZFPs can also be used as models for design and/or assources of zinc finger sequences for modified plant ZFPs. See, e.g., WO96/32475. Other suitable ZFPs are known to those of skill in the art andare described herein. The documents cited herein also disclose methodsof assessing binding specificity of modified ZFPs.

Optionally, modified plant ZFPs can include one or more residues notpresent in a naturally occurring plant zinc finger such as can beobtained by, for example, design and/or selection. For example, one ormore sequence in the alpha-helical region, particularly residuesinvolved in target-recognition (e.g., amino acids −1, +2, +3 and +6),can be altered with respect to a naturally occurring plant ZFP. Anyrecognition sequence can be chosen, for example, by selecting residuesknown to bind to certain target sequences, determined as describedherein and in the references cited herein.

Sequences from any ZFP that is used in the methods described herein canbe altered by mutagenesis, substitution, insertion and/or deletion ofone or more residues so that the non-recognition plant-derived residuesdo not correspond exactly to the zinc finger from which they arederived. Preferably, at least 75% of the modified plant ZFP residueswill correspond to those of the plant sequences, more often 90%, andmost preferably greater than 95%.

In general, modified plant ZFPs are produced by a process of analysis ofplant sequences, for example those sequences that are publicly availableon any number of databases. Three-dimensional modeling can be used, butis not required. Typically, plant sequences are selected for theirhomology to non-plant ZFPs, for example, by selecting plant ZFPs thatmost closely resemble the chosen non-plant ZFP scaffold (e.g., a C₃Hstructures and/or C₂H₂ ZFP structure such as Sp-1 or Sp-1 consensus) andbinding mode. The plant sequences are then assembled in a non-plantbinding mode structure, for instance as three zinc fingers separated byshort linkers, as are present in non-plant ZFPs. Thus, the process ofobtaining a modified plant ZFP with a predetermined binding specificitycan begin by analysis of naturally occurring plant ZFPs.

Once selected plant sequences have been organized and assembled toreflect a non-plant structure, alterations in the recognition residues(i.e., positions −1 to +6 of the alpha helix) can be made so as toconfer a desired binding specificity, for example as described inco-owned WO 00/42219; WO 00/41566; as well as U.S. Pat. Nos. 5,789,538;6,007,408; 6,013,453; 6,140,081 and 6,140,466; and PCT publications WO95/19431, WO 98/54311, WO 00/23464; WO 00/27878; WO98/53057; WO98/53058;WO98/53059; and WO98/53060.

In other embodiments, one or more residues, for example recognitionresidues, can be derived from non-plant sources and inserted into themodified plant ZFP structure. In particular, non-plant sequences thathave previously been shown to bind to specific target sequences can beincorporated into the modified plant ZFP to provide the desired bindingspecificity. Thus, the modified plant ZFPs can include, one or morenon-plant derived residues involved in DNA binding where these bindingresidues have been designed and/or selected to recognize a particulartarget site, for example as described.

In certain embodiments, modified plant ZFPs, as disclosed herein,contain additional modifications in their zinc fingers, for example, asdescribed in applications of which the benefit is claimed herein. Suchadditional modifications include, for example, substitution of azinc-coordinating amino acid residue (i.e., cysteine and/or histidine)with a different amino acid. A modified ZFP of this type can include anynumber of zinc finger components, and, in one embodiment, contains threezinc fingers. Typically, the C-terminal-most (e.g., third) finger of theZFP is substituted in one or more zinc-coordinating residues. The otherfingers of the protein can be naturally occurring zinc fingercomponents, modified plant components, canonical C₂H₂ fingers orcombinations of these components.

Also included herein are nucleic acids encoding a ZFP comprising atleast one modified plant zinc finger as described herein.

B. Linkage

Two or more zinc finger proteins can be linked to have a target sitespecificity that is, to a first approximation, the aggregate of that ofthe component zinc finger proteins. For example, a first modified plantzinc finger protein having first, second and third component fingersthat respectively bind to XXX, YYY and ZZZ can be linked to a secondmodified plant zinc finger protein having first, second and thirdcomponent fingers with binding specificities, AAA, BBB and CCC. Thebinding specificity of the combined first and second proteins is thus5′-CCCBBBAAANZZZYYYXXX-3′ (SEQ ID NO:4), where N indicates a shortintervening region (typically 0-5 bases of any type). In this situation,the target site can be viewed as comprising two target segmentsseparated by an intervening segment.

Linkage of zinc fingers and zinc finger proteins can be accomplishedusing any of the following peptide linkers:

TGEKP (SEQ ID NO: 5) Liu et al. (1997) Proc. Natl. Acad. Sci. USA 94:5525–5530. (G₄S)_(n) (SEQ ID NO: 6) Kim et al. (1996) Proc. Natl. Acad.Sci. USA 93: 1156–1160. GGRRGGGS (SEQ ID NO: 7) LRQRDGERP (SEQ ID NO: 8)LRQKDGGGSERP (SEQ ID NO: 9) LRQKD(G₃S)₂ERP. (SEQ ID NO: 10)

Alternatively, flexible linkers can be rationally designed usingcomputer programs capable of modeling both DNA-binding sites and thepeptides themselves, or by phage display methods. In a furthervariation, non-covalent linkage can be achieved by fusing two zincfinger proteins with domains promoting heterodimer formation of the twozinc finger proteins. For example, one zinc finger protein can be fusedwith fos and the other with jun (see Barbas et al., WO 95/119431).Alternatively, dimerization interfaces can be obtained by selection.See, for example, Wang et al. (1999) Proc. Natl. Acad. Sci. USA96:9568-9573.

C. Fusion Molecules

The modified plant zinc finger proteins described herein can also beused in the design of fusion molecules that facilitate regulation ofgene expression, particularly in plants. Thus, in certain embodiments,the compositions and methods disclosed herein involve fusions between atleast one of the zinc finger proteins described herein (or functionalfragments thereof) and one or more functional domains (or functionalfragments thereof), or a polynucleotide encoding such a fusion. Thepresence of such a fusion molecule in a cell allows a functional domainto be brought into proximity with a sequence in a gene that is bound bythe zinc finger portion of the fusion molecule. The transcriptionalregulatory function of the functional domain is then able to act on thegene, by, for example, modulating expression of the gene.

In certain embodiments, fusion proteins comprising a modified plant zincfinger DNA-binding domain and a functional domain are used formodulation of endogenous gene expression as described, for example, inco-owned PCT WO 00/41566. Modulation includes repression and activationof gene expression; the nature of the modulation generally depending onthe type of functional domain present in the fusion protein. Anypolypeptide sequence or domain capable of influencing gene expression(or functional fragment thereof) that can be fused to a DNA-bindingdomain, is suitable for use.

An exemplary functional domain for fusing with a ZFP DNA-binding domain,to be used for repressing gene expression, is a KRAB repression domainfrom the human KOX-1 protein (see, e.g., Thiesen et al., New Biologist2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA 91,4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914 (1994);Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518 (1994).Another suitable repression domain is methyl binding domain protein 2B(MBD-2B) (see, also Hendrich et al. (1999) Mamm Genome 10:906-912 fordescription of MBD proteins). Another useful repression domain is thatassociated with the v-ErbA protein. See, for example, Damm, et al.(1989) Nature 339:593-597; Evans (1989) Int. J. Cancer Suppl. 4:26-28;Pain et al. (1990) New Biol. 2:284-294; Sap et al. (1989) Nature340:242-244; Zenke et al. (1988) Cell 52:107-119; and Zenke et al.(1990) Cell 61:1035-1049. Additional exemplary repression domainsinclude, but are not limited to, thyroid hormone receptor (TR), SID,MBD1, MBD2, MBD3, MBD4, MBD-like proteins, members of the DNMT family(e.g., DNMT1, DNMT3A, DNMT3B), Rb, MeCP1 and MeCP2. See, for example,Zhang et al. (2000) Ann Rev Physiol 62:439-466; Bird et al. (1999) Cell99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999)Cell 99:447-450; and Robertson et al. (2000) Nature Genet. 25:338-342.Additional exemplary repression domains include, but are not limited to,ROM2 and AtHD2A. See, for example, Chern et al. (1996) Plant Cell8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Seifpalet al., EMBO J. 11, 4961-4968 (1992)).

Additional exemplary activation domains include, but are not limited to,p300, CBP, PCAF, SRC1 PvALF, and ERF-2. See, for example, Robyr et al.(2000) Mol. Endocrinol. 14:329-347; Collingwood et al. (1999) J. Mol.Endocrinol. 23:255-275; Leo et al. (2000) Gene 245:1-11;Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89; McKenna etal. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik et al. (2000)Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999) Curr. Opin.Genet. Dev. 9:499-504. Additional exemplary activation domains include,but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8,CPRF1, CPRF4, MYC-RP/GP, and TRAB1. See, for example, Ogawa et al.(2000) Gene 245:21-29; Okanami et al. (1996) Genes Cells 1:87-99; Goffet al. (1991) Genes Dev. 5:298-309; Cho et al. (1999) Plant Mol. Biol.40:419-429; Ulmason et al. (1999) Proc. Natl. Acad. Sci. USA96:5844-5849; Sprenger-Haussels et al. (2000) Plant J. 22:1-8; Gong etal. (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. (1999) Proc. Natl.Acad. Sci. USA 96:15,348-15,353.

Additional functional domains are disclosed, for example, in co-owned WO00/41566. Further, insulator domains, chromatin remodeling proteins suchas ISWI-containing domains and/or methyl binding domain proteinssuitable for use in fusion molecules are described, for example, inco-owned International Publications WO 01/83793 and PCT/US01/42377.

In additional embodiments, targeted remodeling of chromatin, asdisclosed, for example, in co-owned International Publication WO01/83793, can be used to generate one or more sites in plant cellchromatin that are accessible to the binding of a functionaldomain/modified plant ZFP fusion molecule.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well known to those of skill in the art. Fusionmolecules comprise a modified plant ZFP binding domain and, for example,a transcriptional activation domain, a transcriptional repressiondomain, a component of a chromatin remodeling complex, an insulatordomain or a functional fragment of any of these domains. In certainembodiments, fusion molecules comprise a modified plant zinc fingerprotein and at least two functional domains (e.g., an insulator domainor a methyl binding protein domain and, additionally, a transcriptionalactivation or repression domain). Fusion molecules also optionallycomprise a nuclear localization signal (such as, for example, that fromthe SV40 T-antigen or the maize Opaque-2 NLS) and an epitope tag (suchas, for example, FLAG or hemagglutinin). Fusion proteins (and nucleicacids encoding them) are designed such that the translational readingframe is preserved among the components of the fusion.

The fusion molecules disclosed herein comprise a modified plant zincfinger binding protein that binds to a target site. In certainembodiments, the target site is present in an accessible region ofcellular chromatin. Accessible regions can be determined as described inco-owned International Publications WO 01/83751 and WO 01/83732. If thetarget site is not present in an accessible region of cellularchromatin, one or more accessible regions can be generated as describedin co-owned International Publication WO 01/83793. In additionalembodiments, one or more modified plant zinc finger components of afusion molecule are capable of binding to cellular chromatin regardlessof whether its target site is in an accessible region or not. Forexample, a ZFP as disclosed herein can be capable of binding to linkerDNA and/or to nucleosomal DNA. Examples of this type of “pioneer” DNAbinding domain are found in certain steroid receptors and in hepatocytenuclear factor 3 (HNF3). Cordingley et al. (1987) Cell 48:261-270; Pinaet al. (1990) Cell 60:719-731; and Cirillo et al. (1998) EMBO J.17:244-254.

Methods of gene regulation using a functional domain, targeted to aspecific sequence by virtue of a fused DNA binding domain, can achievemodulation of gene expression. Genes so modulated can be endogenousgenes or exogenous genes. Modulation of gene expression can be in theform of repression (e.g., repressing expression of exogenous genes, forexample, when the target gene resides in a pathological infectingmicroorganism, or repression of an endogenous gene of the subject, suchas an oncogene or a viral receptor, that contributes to a diseasestate). As described herein, repression of a specific target gene can beachieved by using a fusion molecule comprising a modified plant zincfinger protein and a functional domain.

Alternatively, modulation can be in the form of activation, ifactivation of a gene (e.g., a tumor suppressor gene or a transgene) canameliorate a disease state. In this case, a cell is contacted with anyof the fusion molecules described herein, wherein the modified zincfinger portion of the fusion molecule is specific for the target gene.The target gene can be an exogenous gene such as, for example, atransgene, or it can be an endogenous cellular gene residing in cellularchromatin. The functional domain (e.g., insulator domain, activationdomain, etc.) enables increased and/or sustained expression of thetarget gene.

For any such applications, the fusion molecule(s) and/or nucleic acidsencoding one or more fusion molecules can be formulated with anacceptable carrier, to facilitate introduction into and/or expression inplant cells, as is known to those of skill in the art.

Polynucleotide and Polypeptide Delivery

The compositions described herein can be provided to the target cell invitro or in vivo. In addition, the compositions can be provided aspolypeptides, polynucleotides or combination thereof.

A. Delivery of Polynucleotides

In certain embodiments, the compositions are provided as one or morepolynucleotides. Further, as noted above, a modified plant zinc fingerprotein-containing composition can be designed as a fusion between apolypeptide zinc finger and a functional domain that is encoded by afusion nucleic acid. In both fusion and non-fusion cases, the nucleicacid can be cloned into intermediate vectors for transformation intoprokaryotic or eukaryotic (e.g., plant) cells for replication and/orexpression. Intermediate vectors for storage or manipulation of thenucleic acid or production of protein can be prokaryotic vectors, (e.g.,plasmids), shuttle vectors, insect vectors, or viral vectors forexample. A nucleic acid encoding a modified plant zinc finger proteincan also cloned into an expression vector, for administration to abacterial cell, fungal cell, protozoal cell, plant cell, or animal cell,preferably a plant cell.

To obtain expression of a cloned nucleic acid, it is typically subclonedinto an expression vector that contains a promoter to directtranscription. Suitable bacterial and eukaryotic promoters are wellknown in the art and described, e.g., in Sambrook et al., supra; Ausubelet al., supra; and Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990). Bacterial expression systems are available in, e.g., E.coli, Bacillus sp., and Salmonella. Palva et al. (1983) Gene 22:229-235.Kits for such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available, for example, fromInvitrogen, Carlsbad, Calif. and Clontech, Palo Alto, Calif.

Plant expression vectors and reporter genes are also generally known inthe art. (See, e.g., Gruber et al. (1993) in Methods of Plant MolecularBiology and Biotechnology, CRC Press.) Such systems include in vitro andin vivo recombinant DNA techniques, and any other synthetic or naturalrecombination. (See, e.g., Transgenic Plants: A Production System forIndustrial and Pharmaceutical Proteins, Owen and Pen eds., John Wiliey &Sons, 1996; Transgenic Plants, Galun and Breiman eds, Imperial CollegePress, 1997; Applied Plant Biotechnology, Chopra, Malik, and Bhat eds.,Science Publishers, Inc., 1999.)

The promoter used to direct expression of the nucleic acid of choicedepends on the particular application. For example, a strongconstitutive promoter is typically used for expression and purification.In contrast, when a protein is to be used in vivo, either a constitutiveor an inducible promoter is used, depending on the particular use of theprotein. In addition, a weak promoter can be used, when low butsustained levels of protein are required. The promoter typically canalso include elements that are responsive to transactivation, e.g.,hypoxia response elements and small molecule control systems such astet-regulated systems and the RU-486 system. See, e.g., Gossen et al.(1992) Proc. Natl. Acad. Sci USA 89:5547-5551; Oligino et al. (1998)Gene Ther. 5:491-496; Wang et al. (1997) Gene Ther. 4:432-441; Neeringet al. (1996) Blood 88:1147-1155; and Rendahl et al. (1998) Nat.Biotechnol. 16:757-761.

Promoters suitable for use in plant expression systems include, but arenot limited to, viral promoters such as the 35S RNA and 19S RNApromoters of cauliflower mosaic virus (CaMV) (Brisson et al. (1984)Nature 310:511-514, Example 1); the coat protein promoter of TMV(Takamatsu et al. (1987) EMBO J. 6:307-311); plant promoters such as thesmall subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680;Broglie et al. (1984) Science 224:838-843; plant heat shock promoters,e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al. (1986) Cell. Biol.6:559-565) may be used. Other examples of promoters that may be used inexpression vectors comprising nucleotides encoding modified plant ZFPsinclude the promoter for the small subunit of ribulose-1,5-bis-phosphatecarboxylase; promoters from tumor-inducing plasmids of Agrobacteriumtumefaciens, such as the RUBISCO nopaline synthase (NOS) and octopinesynthase promoters; bacterial T-DNA promoters such as mas and ocspromoters; or the figwort mosaic virus 35S promoter.

In a preferred embodiment, the modified plant ZFP polynucleotidesequence is under the control of the cauliflower mosaic virus (CaMV) 35Spromoter (Example 3). The caulimorvirus family has provided a number ofexemplary promoters for transgene expression in plants, in particular,the (CaMV) 35S promoter. (See, e.g., Kay et al. (1987) Science236:1299.) Additional promoters from this family such as the figwortmosaic virus promoter, the Commelina yellow mottle virus promoter, andthe rice tungro bacilliform virus promoter have been described in theart, and may also be used in the methods and compositions disclosedherein. (See, e.g., Sanger et al. (1990) Plant Mol. Biol. 14:433-443;Medberry et al. (1992) Plant Cell 4:195-192; Yin and Beachy (1995) PlantJ. 7:969-980.)

The promoters may be modified, if desired, to affect their controlcharacteristics. For example, the CaMV 35S promoter may be ligated tothe portion of the RUBISCO gene that represses the expression of RUBISCOin the absence of light, to create a promoter that is active in leaves,but not in roots. The resulting chimeric promoter may be used asdescribed herein. Constitutive plant promoters such as actin andubiquitin, having general expression properties known in the art may beused to express modified plant ZFPs. (See, e.g., McElroy et al. (1990)Plant Cell 2:163-171; Christensen et al. (1992) Plant Mol. Biol.18:675-689.)

Additionally, depending on the desired tissue, expression may betargeted to the endosperm, aleurone layer, embryo (or its parts asscutellum and cotyledons), pericarp, stem, leaves tubers, roots, etc.Examples of known tissue-specific promoters include the tuber-directedclass I patatin promoter, the promoters associated with potato tuberADPGPP genes, the soybean promoter of β-conglycinin (7S protein) whichdrives seed-directed transcription, and seed-directed promoters from thezein genes of maize endosperm. (See, e.g., Bevan et al., 1986, NucleicAcids Res. 14: 4625-38; Muller et al., 1990, Mol. Gen. Genet. 224:136-46; Bray, 1987, Planta 172: 364-370; Pedersen et al., 1982, Cell 29:1015-26.) Additional seed-specific promoters include the phaseolin andnapin promoters.

In addition to a promoter, an expression vector typically contains atranscription unit or expression cassette that contains additionalelements required for the expression of the nucleic acid in host cells,either prokaryotic or eukaryotic. A typical expression cassette thuscontains a promoter operably linked, e.g., to the nucleic acid sequence,and signals required, e.g., for efficient polyadenylation of thetranscript, transcriptional termination, ribosome binding, and/ortranslation termination. Additional elements of the cassette mayinclude, e.g., enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe resulting ZFP polypeptide, e.g., expression in plants.

In addition, the recombinant constructs may include plant-expressibleselectable or screenable marker genes for isolating, identifying ortracking of plant cells transformed by these constructs. Selectablemarkers include, but are not limited to, genes that confer antibioticresistances (e.g., resistance to kanamycin or hygromycin) or herbicideresistance (e.g., resistance to sulfonylurea, phosphinothricin, orglyphosate). Screenable markers include, but are not limited to, thegenes encoding beta-glucuronidase (Jefferson (1987) Plant Molec Biol.Rep 5:387-405), luciferase (Ow et al. (1986) Science 234:856-859), andthe B and C1 gene products that regulate anthocyanin pigment production(Goff et al. (1990) EMBO J. 9:2517-2522).

Other elements that are optionally included in expression vectors alsoinclude a replicon that functions in E. coli (or in the prokaryotichost, if other than E. coli), a selective marker that functions in aprokaryotic host, e.g., a gene encoding antibiotic resistance, to permitselection of bacteria that harbor recombinant plasmids, and uniquerestriction sites in nonessential regions of the vector to allowinsertion of recombinant sequences.

Standard transfection methods can be used to produce bacterial,mammalian, yeast, insect, other cell lines or, preferably, plants thatexpress large quantities of modified plant zinc finger proteins, whichcan be purified, if desired, using standard techniques. See, e.g.,Colley et al. (1989) J. Biol. Chem. 264:17619-17622; and Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed.) 1990. Transformation of non-plant eukaryotic cells and prokaryoticcells are performed according to standard techniques. See, e.g.,Morrison (1977) J. Bacteriol. 132:349-351; Clark-Curtiss et al. (1983)in Methods in Enzymology 101:347-362 (Wu et al., eds).

Transformation systems for plants as also known. (See, e.g., Weissbach &Weissbach, Methods for Plant Molecular Biology, Academic Press, NY,Section VIII, pp. 421-463 (1988); Grierson & Corey, Plant MolecularBiology, 2d Ed., Blackie, London, Ch. 7-9 (1988).) For example,Agrobacterium is often successfully employed to introduce nucleic acidsinto plants. Such transformation preferably uses binary AgrobacteriumT-DNA vectors which can be used to transform dicotyledonous plants,monocotyledonous plants and plant cells (Bevan (1984) Nuc. Acid Res.12:8711-8721; Horsch et al. (1985) Science 227:1229-1231; Bevan et al.(1982) Ann. Rev. Genet 16:357-384; Rogers et al. (1986) Methods Enzymol.118:627-641; Hernalsteen et al. (1984) EMBO J 3:3039-3041). Inembodiments that utilize the Agrobacterium system for transformingplants, the recombinant DNA constructs typically comprise at least theright T-DNA border sequence flanking the DNA sequences to be transformedinto the plant cell. In preferred embodiments, the sequences to betransferred are flanked by the right and left T-DNA border sequences.The design and construction of such T-DNA based transformation vectorsare well known to those skilled in the art.

Other gene transfer and transformation methods include, but are notlimited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA (seePaszkowski et al. (1984) EMBO J. 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276);electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505); microinjection, silicon carbide mediated DNA uptake(Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), microprojectilebombardment (see Klein et al. (1983) Proc. Nat. Acad. Sci. USA85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618);direct gene transfer, in vitro protoplast transformation, plantvirus-mediated transformation, liposome-mediated transformation, andballistic particle acceleration (See, e.g., Paszkowski et al. (1984)EMBO J. 3:2717-2722; U.S. Pat. Nos. 4,684,611; 4,407,956; 4,536,475;Crossway et al., (1986) Biotechniques 4:320-334; Riggs et al (1986)Proc. Natl. Acad. Sci USA 83:5602-5606; Hinchee et al. (1988)Biotechnology 6:915-921; U.S. Pat. No. 4,945,050.)

A wide variety of host cells, plants and plant cell systems can be used,including, but not limited to, those monocotyledonous and dicotyledonousplants, such as crops including grain crops (e.g., wheat, maize, rice,millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry,orange), forage crops (e.g., alfalfa), root vegetable crops (e.g.,carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce,spinach); flowering plants (e.g., petunia, rose, chrysanthemum),conifers and pine trees (e.g., pine fir, spruce); plants used inphytoremediation (e.g., heavy metal accumulating plants); oil crops(e.g., sunflower, rape seed) and plants used for experimental purposes(e.g., Arabidopsis).

Modified plant ZFPs and the resulting gene product the ZFP modulates canalso be produced from seed by way of seed-based production techniquesusing, for example, canola, corn, soybeans, rice and barley seed, andthe modified plant ZFP, and/or sequences encoding it, can be recoveredduring seed germination. See, e.g., PCT Publication Numbers WO 9940210;WO 9916890; WO 9907206; U.S. Pat. No. 5,866,121; and U.S. Pat. No.5,792,933; and all references cited therein.

B. Delivery of Polypeptides

In additional embodiments, modified plant ZFPs or fusion proteinscomprising modified plant ZFPs are administered directly to target plantcells. In certain in vitro situations, the target cells are cultured ina medium containing a fusion protein comprising one or more functionaldomains fused to one or more of the modified plant ZFPs describedherein. An important factor in the administration of polypeptidecompounds in plants is ensuring that the polypeptide has the ability totraverse a cell wall. However, proteins, viruses, toxins, ballisticmethods and the like have the ability to translocate polypeptides acrossa plant cell wall.

For example, “plasmodesmata” is the term given to explain cell-to-celltransport of endogenous and viral proteins and ribonucleoproteincomplexes (RNPCs) in plants. Examples of viruses which can be linked toa modified plant zinc finger polypeptide (or fusion containing the same)for facilitating its uptake into plant cells include, tobacco mosaicvirus (Oparka et al. (1997) Plant J. 12:781-789; rice phloem thioredoxin(Ishiwatari et al. (1998) Planta 205:12-22); potato virus X (Cruz et al.(1998) Plant Cell 10:495-510) and the like. Other suitable chemicalmoieties that provide enhanced cellular uptake can also be linked,either covalently or non-covalently, to the ZFPs. Toxin molecules alsohave the ability to transport polypeptides across cell walls.

Particle-mediated delivery techniques (e.g., ballistic injection) asdescribed above regarding nucleic acids can also be used to introducepolypeptides into a plant cell.

Applications

The modified plant zinc finger proteins and fusion molecules disclosedherein, and expression vectors encoding these polypeptides, can be usedto modulate the expression of, or the action of, any plant endogenous orexogenous gene or gene product. In such applications, modified plantZFP-containing compositions can be administered directly to a plant,e.g., to facilitate the modulation of gene expression. Preferably, themodulated gene is endogenous, for example a gene involved in growth,development, morphology, seed or fruit-bearing ability and the like. Thegene product itself may be isolated and, accordingly, modulation ofendogenous plant genes can be achieved using plant-derived sequences.

Accordingly, expression of any gene in any organism, for example plantsor fungi, can be modulated using the methods and compositions disclosedherein, including therapeutically relevant genes, genes of infectingmicroorganisms, viral genes, and genes whose expression is modulated inthe processes of drug discovery and/or target validation. Such genesinclude, but are not limited to, Wilms' third tumor gene (WT3), vascularendothelial growth factors (VEGFs), VEGF receptors (e.g., flt and flk)CCR-5, low density lipoprotein receptor (LDLR), estrogen receptor,HER-2/neu, BRCA-1, BRCA-2, phosphoenolpyruvate carboxykinase (PEPCK),CYP7, fibrinogen, apolipoprotein A (ApoA), apolipoprotein B (ApoB),renin, phosphoenolpyruvate carboxykinase (PEPCK), CYP7, fibrinogen,nuclear factor κB (NF-κB), inhibitor of NF-κB (I-κB), tumor necrosisfactors (e.g., TNF-α, TNF-β), interleukin-1 (IL-1), FAS (CD95), FASligand (CD95L), atrial natriuretic factor, platelet-derived factor(PDF), amyloid precursor protein (APP), tyrosinase, tyrosinehydroxylase, β-aspartyl hydroxylase, alkaline phosphatase, calpains(e.g., CAPN10) neuronal pentraxin receptor, adriamycin response protein,apolipoprotein E (apoE), leptin, leptin receptor, UCP-1, IL-1, IL-1receptor, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-15, interleukinreceptors, G-CSF, GM-CSF, colony stimulating factor, erythropoietin(EPO), platelet-derived growth factor (PDGF), PDGF receptor, fibroblastgrowth factor (FGF), FGF receptor, PAF, p16, p19, p53, Rb, p21, myc,myb, globin, dystrophin, eutrophin, cystic fibrosis transmembraneconductance regulator (CFTR), GNDF, nerve growth factor (NGF), NGFreceptor, epidermal growth factor (EGF), EGF receptor, transforminggrowth factors (e.g., TGF-α, TGF-β), fibroblast growth factor (FGF),interferons (e.g., IFN-α, IFN-β and IFN-γ), insulin-related growthfactor-1 (IGF-1), angiostatin, ICAM-1, signal transducer and activatorof transcription (STAT), androgen receptors, e-cadherin, cathepsins(e.g., cathepsin W), topoisomerase, telomerase, bcl, bcl-2, Bax, TCell-specific tyrosine kinase (Lck), p38 mitogen-activated proteinkinase, protein tyrosine phosphatase (hPTP), adenylate cyclase,guanylate cyclase, α7 neuronal nicotinic acetylcholine receptor,5-hydroxytryptamine (serotonin)-2A receptor, transcription elongationfactor-3 (TEF-3), phosphatidylcholine transferase, ftz, PTI-1,polygalacturonase, EPSP synthase, FAD2-1, Δ-9 desaturase, Δ-12desaturase, Δ-15 desaturase, acetyl-Coenzyme A carboxylase, acyl-ACPthioesterase, ADP-glucose pyrophosphorylase, starch synthase, cellulosesynthase, sucrose synthase, fatty acid hydroperoxide lyase, andperoxisome proliferator-activated receptors, such as PPAR-γ2.

Expression of human, mammalian, bacterial, fungal, protozoal, Archaeal,plant and viral genes can be modulated; viral genes include, but are notlimited to, hepatitis virus genes such as, for example, HBV-C, HBV-S,HBV-X and HBV-P; and

HIV genes such as, for example, tat and rev. Modulation of expression ofgenes encoding antigens of a pathogenic organism can be achieved usingthe disclosed methods and compositions.

In other embodiments, the modulated gene can be exogenous, for example,a transgene that has been inserted into the plant. Techniques forgenerating transgenic plants are known in the art (see, e.g., Swain W F(1991) TIBTECH 9: 107-109; Ma J K C et al. (1994) Eur J Immunology24:131-138; Hiatt A et al. (1992) FEBS Letters 307:71-75; Hein M B etal. (1991) Biotechnology Progress 7: 455-461; Duering K (1990) PlantMolecular Biology 15: 281-294). As with endogenous genes, the modifiedplant ZFP (or fusion polypeptides comprising the modified plant ZFPsdescribed herein) can then modulate expression of a transgene, forexample to produce a protein product of interest, without the need forregulatory molecules derived primarily from non-plant (e.g., animal)sources.

Accordingly, the compositions and methods disclosed herein can be usedto facilitate a number of processes involving transcriptional regulationin plants. These processes include, but are not limited to,transcription, replication, recombination, repair, integration,maintenance of telomeres, processes involved in chromosome stability anddisjunction, and maintenance and propagation of chromatin structures.The methods and compositions disclosed herein can be used to affect anyof these processes, as well as any other process that can be influencedby ZFPs or ZFP fusions.

Additional exemplary applications for modulation of gene expression inplant cells using modified plant ZFPs include, for example, theoptimization of crop traits affecting nutritional value, yield, stresstolerance, pathogen resistance, and resistance to agrochemicals (e.g.insecticides and/or herbicides). In addition, targeted gene regulationcan be used to study gene function in plants, and to adapt plants foruse as biological factories for the production of pharmaceuticalcompounds or industrial chemicals.

In preferred embodiments, one or more of the molecules described hereinare used to achieve targeted activation or repression of geneexpression, e.g., based upon the target site specificity of the modifiedplant ZFP. In another embodiment, one or more of the molecules describedherein are used to achieve reactivation of a gene, for example adevelopmentally silenced gene; or to achieve sustained activation of atransgene. A modified plant ZFP can be targeted to a region outside ofthe coding region of the gene of interest and, in certain embodiments,is targeted to a region outside of known regulatory region(s) of thegene. In these embodiments, additional molecules, exogenous and/orendogenous, can optionally be used to facilitate repression oractivation of gene expression. The additional molecules can also befusion molecules, for example, fusions between a ZFP and a functionaldomain such as an activation or repression domain. See, for example,co-owned WO 00/41566.

In other applications, modified plant ZFPs and other DNA- and/orRNA-binding proteins are used in diagnostic methods forsequence-specific detection of target nucleic acid in a sample. Forexample, modified plant ZFPs can be used to detect variant allelesassociated with a phenotype in a plant. As an example, modified plantZFPs can be used to detect the presence of particular mRNA species orcDNA in a complex mixtures of mRNAs or cDNAs. As a further example,modified plant ZFPs can be used to quantify the copy number of a gene ina sample. A suitable format for performing diagnostic assays employsmodified plant ZFPs linked to a domain that allows immobilization of theZFP on a solid support such as, for example, a microtiter plate or anELISA plate. The immobilized ZFP is contacted with a sample suspected ofcontaining a target nucleic acid under conditions in which bindingbetween the modified ZFP and its target sequence can occur. Typically,nucleic acids in the sample are labeled (e.g., in the course of PCRamplification). Alternatively, unlabelled nucleic acids can be detectedusing a second labeled probe nucleic acid. After washing, bound, labelednucleic acids are detected. Labeling can be direct (i.e., the probebinds directly to the target nucleic acid) or indirect (i.e., probebinds to one or more molecules which themselves bind to the target).Labels can be, for example, radioactive, fluorescent, chemiluminescentand/or enzymatic.

Modified plant ZFPs, as disclosed herein, can also be used in assaysthat link phenotype to the expression of particular genes. Currentmethodologies for determination of gene function rely primarily uponeither over-expressing a gene of interest or removing a gene of interestfrom its natural biological setting, and observing the effects. Thephenotypic effects resulting from over-expression or knockout are theninterpreted as an indication of the role of the gene in the biologicalsystem. Up- or down-regulation of gene expression using one or moremodified plant ZFPs obviates the necessity of generating transgenicplants for use in these types of assay.

All references cited herein are hereby incorporated by reference intheir entirety for all purposes.

The following examples are presented as illustrative of, but notlimiting, the claimed subject matter.

EXAMPLES Example 1 Production of Modified Plant Zinc Finger BindingProteins

This example describes a strategy to select amino acid sequences forplant zinc finger backbones from among existing plant zinc fingersequences, and subsequent conceptual modification of the selected plantzinc finger amino acid sequences to optimize their DNA binding ability.Oligonucleotides used in the preparation of polynucleotides encodingproteins containing these zinc fingers in tandem array are thendescribed.

A. Selection of Plant Zinc Finger Backbones

A search was conducted for plant zinc fingers whose backbone sequences(i.e., the portion of the zinc finger outside of the −1 through +6portion of the recognition helix) resembled that of the SP-1 consensussequence described by Berg (1992) Proc. Natl. Acad. Sci. USA89:11,109-11,110. The sequences selected included the two conservedcysteine residues, a conserved basic residue (lysine or arginine)located two residues to the C-terminal side of the second (i.e.C-terminal) cysteine, a conserved phenylalanine residue located tworesidues to the C-terminal side of the basic residue, the two conservedhistidine residues, and a conserved arginine residue located tworesidues to the C-terminal side of the first (i.e., N-terminal)conserved histidine. The amino acid sequences of these selected plantzinc finger backbones (compared to the SP-1 consensus sequence) areshown below, with conserved residues shown in bold and X referring toresidues located at positions −1 through +6 in the recognition helix(which will differ among different proteins depending upon the targetsequence):

(SEQ ID NO: 11) SP-1 consensus:       YKCPECGKSFSXXXXXXXHQRTHTGEKP (SEQID NO: 12) F1: KKKSKGHECPICFRVFKXXXXXXXHKRSHTGEKP (SEQ ID NO: 13) F2      YKCTVCGKSFSXXXXXXXHKRLHTGEKP (SEQ ID NO: 14) F3      FSCNYCQRKFYXXXXXXXHVRIH             −5  −1    5

The first finger (F1) was chosen because it contained a basic sequenceN-terminal to the finger that is also found adjacent to the first fingerof SP-1. The finger denoted F1 is a Petunia sequence, the F2 and F3fingers are Arabidopsis sequences.

B. Modification of Plant Zinc Finger Backbones

Two of the three plant zinc fingers (F1 and F3, above) were modified sothat their amino acid sequences more closely resembled the sequence ofSP-1, as follows. (Note that the sequence of SP-1 is different from thesequence denoted “SP-1 consensus.”) In F3, the Y residue at position −2was converted to a G, and the sequence QNKK (SEQ ID NO:15) was added tothe C-terminus of F3. The QNKK sequence is present C-terminal to thethird finger of SP-1, and permits greater flexibility of that finger,compared to fingers 1 and 2, which are flanked by the helix-cappingsequence T G E K/R K/P (SEQ ID NO:16). Such flexibility can bebeneficial when the third finger is modified to contain a non-C₂H₂structure. ** Finally, several amino acids were removed from theN-terminus of F1. The resulting zinc finger backbones had the followingsequences:

KSKGHECPIC FRVFKXXXXXXXHKR SHTGEKP (SEQ ID NO: 17)     YKCTVCGKSFSXXXXXXXHKR LHTGEKP (SEQ ID NO: 18)     FSCNYC QRKFG XXXXXXXHVRIHQNKK(SEQ ID NO: 19)

Amino acid residues denoted by X, present in the recognition portion ofthese zinc fingers, are designed or selected depending upon the desiredtarget site, according to methods disclosed, for example, in co-owned WO00/41566 and WO 00/42219, and/or references cited supra.

C. Nucleic Acid Sequences Encoding Backbones for Modified Plant ZFPs

The following polynucleotide sequences are used for design of athree-finger plant ZFP that contains the F1, F2 and F3 backbonesdescribed above. Polynucleotides encoding multi-finger ZFPs are designedaccording to an overlapping oligonucleotide method as described in, forexample, co-owned WO 00/41566 and WO 00/42219. Oligonucleotides H1, H2and H3 (below) comprise sequences corresponding to the reversecomplement of the recognition helices of fingers 1-3 respectively;accordingly, nucleotides denoted by N will vary depending upon thedesired amino acid sequences of the recognition helices, which, in turn,will depend upon the nucleotide sequence of the target site.Oligonucleotides PB1, PB2 and PB3 encode the beta-sheet portions of thezinc fingers, which are common to all constructs. Codons used frequentlyin Arabidopsis and E. coli were selected for use in theseoligonucleotides.

H1: (SEQ ID NO: 20) 5′-CTC ACC GGT GTG AGA ACG CTT GTG NNN NNN NNN NNNNNN NNN NNN CTT GAA AAC ACG GAA-3′ H2: (SEQ ID NO: 21) 5′-TTC ACC AGTATG AAG ACG CTT ATG NNN NNN NNN NNN NNN NNN NNN AGA AAA AGA CTT ACC-3′H3: (SEQ ID NO: 22) 5′-CTT CTT GTT CTG GTG GAT ACG CAC GTG NNN NNN NNNNNN NNN NNN NNN ACC GAA CTT ACG CTG-3′ PB1: (SEQ ID NO: 23)5′-AAGTCTAAGGGTCACGAGTGCCCAATCTGCTTCCGTGTTTTCAAG- 3′ PB2: (SEQ ID NO:24) 5′-TCTCACACCGGTGAGAAGCCATACAAGTGCACTGTTTGTGGTAAGTC TTTTTCT-3′ PB3:(SEQ ID NO: 25) 5′-CTTCATACTGGTGAAAAGCCATTCTCTTGCAACTACTGCCAGCGTAAGTTCGGT-3′

Briefly, these six oligonucleotides are annealed and amplified bypolymerase chain reaction. The initial amplification product isreamplified using primers that are complementary to the initialamplification product and that also contain 5′ extensions containingrestriction enzyme recognition sites, to facilitate cloning. The secondamplification product is inserted into a vector containing, for example,one or more functional domains, nuclear localization sequences, and/orepitope tags. See, for example, co-owned WO 00/41566 and WO 00/42219.

Example 2 Construction of a Polynucleotide Encoding a Modified PlantZinc Finger Protein for Binding to a Predetermined Target Sequence

A modified plant zinc finger protein was designed to recognize thetarget sequence 5′-GAGGGGGCG-3′ (SEQ ID NO:26). Recognition helixsequences for F1, F2 and F3 were determined, as shown in Table 1, andoligonucleotides corresponding to H1, H2 and H3 above, also includingsequences encoding these recognition helices, were used for PCR assemblyas described above.

TABLE 1 Finger Target Helix sequence Nucleotide sequence for PCRassembly F1 GCG RSDELTR 5′CTCACCGGTGTGAGAACGCTTGTGACGGGTCAACT SEQ ID NO:27 CGTCAGAACGCTTGAAAACACGGAA-3′ (SEQ ID NO: 28) F2 GGG RSDHLTR5′TTCACCAGTATGAAGACGCTTATGACGGGTCAAGT SEQ ID NO: 29GGTCAGAACGAGAAAAAGACTTACC-3′ (SEQ ID NO: 30) F3 GAG RSDNLTR5′CTTCTTGTTCTGGTGGATACGCACGTGACGGGTCA SEQ ID NO: 31AGTTGTCAGAACGACCGAACTTACGCTG-3′ (SEQ ID NO: 32)

Subsequent to the initial amplification, a secondary amplification wasconducted, as described above, using the following primers:

(SEQ ID NO: 33) PZF: 5′-CGGGGTACC AGGTAAGTCTAAGGGTCAC (SEQ ID NO: 34)PZR: 5′-GCGCGGATCC ACCCTTCTTGTTCTGGTGGATACG.

PZF includes a KpnI site (underlined) and overlaps the PB1 sequence(overlap indicated in bold). PZR includes a BamHI (underlined) site andoverlaps with H3 (indicated in bold).

The secondary amplification product is digested with Kpn I and Bam HIand inserted into an appropriate vector (e.g., YCF3, whose constructionis described below) to construct an expression vector encoding amodified plant ZFP fused to a functional domain, for modulation of geneexpression in plant cells.

Example 3 Construction of Vectors for Expression of Modified Plant ZFPs

YCF3 was generated as shown schematically in FIG. 1. The startingconstruct was a plasmid containing a CMV promoter, a SV40 nuclearlocalization sequence (NLS), a ZFP DNA binding domain, a HerpesvirusVP16 transcriptional activation domain and a FLAG epitope tag(pSB5186-NVF). This construct was digested with SpeI to remove the CMVpromoter. The larger fragment was gel-purified and self-ligated to makea plasmid termed GF1. GF1 was then digested with KpnI and HindIII,releasing sequences encoding the ZFP domain, the VP16 activation domain,and the FLAG epitope tag, then the larger fragment was ligated to aKpnI/HindIII fragment containing sequences encoding a ZFP binding domainand a VP16 activation domain, named GF2. This resulted in deletion ofsequences encoding the FLAG tag from the construct.

GF2 was digested with BamHI and HindIII, releasing a small fragmentencoding the VP16 activation domain, and the larger fragment waspurified and ligated to a BamHI/HindIII digested PCR fragment containingthe maize C1 activation domain (Goff et al. (1990) EMBO J. 9:2517-2522)(KpnI and HindIII sites were introduced into the PCR fragment throughKpnI and HindIII site-containing primers) to generate NCF1. A PCRfragment containing a Maize Opaque-2 NLS was digested with SpeI/KpnI andligated to the larger fragment from KpnI/SpeI digested NCF1 to produceYCF2. YCF2 was then digested with MluI and SpeI and the larger fragmentwas ligated to an MluI and SpeI digested PCR fragment containing theplant-derived CaMV 35S promoter (MluI and SpeI sites were introducedinto the PCR fragment through MluI or SpeI site containing primers) togenerate the YCF3 vector.

Sequences encoding modified plant ZFP binding domains can be inserted,as KpnI/BamHI fragments, into KpnI/BamHI-digested YCF3 to generateconstructs encoding ZFP-functional domain fusion proteins for modulationof gene expression in plant cells. For example, a series of modifiedplant ZFP domains, described in Example 4 infra, were inserted intoKpnI/BamHI-digested YCF3 to generate expression vectors encodingmodified plant ZFP-activation domain fusion polypeptides that enhanceexpression of the Arabidopsis thaliana GMT gene.

Example 4 Modified Plant ZFP Designs for Regulation of an Arabidopsisthaliana Gamma Tocopherol Methyltransferase (GMT) Gene

Modified plant zinc finger proteins were designed to recognize varioustarget sequences in the Arabidopsis GMT gene (GenBank Accession NumberAAD38271. Table 2 shows the nucleotide sequences of the various GMTtarget sites, and the amino acid sequences of zinc fingers thatrecognize the target sites. Sequences encoding these binding domainswere prepared as described in Example 1 and inserted into YCF3 asdescribed in Example 3.

TABLE 2 ZFP # Target F1 F2 F3 1 GTGGACGAGT RSDNLAR DRSNLTR RSDALTR (SEQID NO: 35) (SEQ ID NO: 36) (SEQ ID NO: 37) (SEQ ID NO: 38) 2 CGGGATGGGTRSDHLAR TSGNLVR RSDHLRE (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 41)(SEQ ID NO: 42) 3 TGGTGGGTGT RSDALTR RSDHLTT RSDHLTT (SEQ ID NO: 43)(SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46) 4 GAAGAGGATT QSSNLARRSDNLAR QSGNLTR (SEQ ID NO: 47) (SEQ ID NO: 48) (SEQ ID NO: 49) (SEQ IDNO: 50) 5 GAGGAAGGGG RSDHLAR QSGNLAR RSDNLTR (SEQ ID NO: 51) (SEQ ID NO:52) (SEQ ID NO: 53) (SEQ ID NO: 54) 6 TGGGTAGTC ERGTLAR QSGSLTR RSDHLTT(SEQ ID NO: 55) (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID NO: 58) 7GGGGAAAGGG RSDHLTQ QSGNLAR RSDHLSR (SEQ ID NO: 59) (SEQ ID NO: 60) (SEQID NO: 61) (SEQ ID NO: 62) 8 GAAGAGGGTG QSSHLAR RSDNLAR QSGNLAR (SEQ IDNO: 63) (SEQ ID NO: 64) (SEQ ID NO: 65) (SEQ ID NO: 66) 9 GAGGAGGATGQSSNLQR RSDNALR RSDNLQR (SEQ ID NO: 67) (SEQ ID NO: 68) (SEQ ID NO: 69)(SEQ ID NO: 70) 10 GAGGAGGAGG RSDNALR RSDNLAR RSDNLTR (SEQ ID NO: 71)(SEQ ID NO: 72) (SEQ ID NO: 73) (SEQ ID NO: 74) 11 GTGGCGGCTG QSSDLRRRSDELQR RSDALTR (SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 77) (SEQ IDNO: 78) 12 TGGGGAGAT QSSNLAR QSGHLQR RSDHLTT (SEQ ID NO: 79) (SEQ ID NO:80) (SEQ ID NO: 81) (SEQ ID NO: 82) 13 GAGGAAGCT QSSDLRR QSGNLAR RSDNLTR(SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID NO: 85) (SEQ ID NO: 86) 14GCTTGTGGCT DRSHLTR TSGHLTT QSSDLTR (SEQ ID NO: 87) (SEQ ID NO: 88) (SEQID NO: 89) (SEQ ID NO: 90) 15 GTAGTGGATG QSSNLAR RSDALSR QSGSLTR (SEQ IDNO: 91) (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 94) 16 GTGTGGGATTQSSNLAR RSDHLTT RSDALTR (SEQ ID NO: 95) (SEQ ID NO: 96) (SEQ ID NO: 97)(SEQ ID NO: 98)

Example 5 Modulation of Expression of an Arabidopsis thaliana GammaTocopherol Methyltransferase (GMT) Gene

Arabidopsis thaliana protoplasts were prepared and transfected withplasmids encoding modified ZFP-activation domain fusion polypeptides.Preparation of protoplasts and polyethylene glycol-mediated transfectionwere performed as described. Abel et al. (1994) Plant Journal 5:421-427.The different plasmids contained the modified plant ZFP binding domainsdescribed in Table 2, inserted as KpnI/BamHI fragments into YCF3.

At 18 hours after transfection, RNA was isolated from transfectedprotoplasts, using an RNA extraction kit from Qiagen (Valencia, Calif.)according to the manufacturer's instructions. The RNA was then treatedwith DNase (RNase-free), and analyzed for GMT mRNA content by real-timePCR (TaqMan®). Table 3 shows the sequences of the primers and probe usedfor TaqMan® analysis. Results for GMT mRNA levels were normalized tolevels of 18S rRNA. These normalized results are shown in FIG. 2 asfold-activation of GMT mRNA levels, compared to protoplasts transfectedwith carrier DNA (denoted “No ZFP” in FIG. 2). The results indicate thatexpression of the GMT gene was enhanced in protoplasts that weretransfected with plasmids encoding fusions between a transcriptionalactivation domain and a modified plant ZFP binding domain targeted tothe GMT gene.

TABLE 3 SEQUENCE GMT forward 5′-AATGATCTCGCGGCTGCT-3′ primer (SEQ ID NO:99) GMT reverse 5′-GAATGGCTGATCCAACGCAT-3′ primer (SEQ ID NO: 100) GMTprobe 5′-TCACTCGCTCATAAGGCTTCCTTCCAAGT-3′ (SEQ ID NO: 101) 18S forward5′-TGCAACAAACCCCGACTTATG-3′ primer (SEQ ID NO: 102) 18S reverse5′-CCCGCGTCGACCTTTTATC-3′ primer (SEQ ID NO: 103) 18S probe5′-AATAAATGCGTCCCTT-3′ (SEQ ID NO: 104)

Although the foregoing methods and compositions have been described indetail for purposes of clarity of understanding, certain modifications,as known to those of skill in the art, can be practiced within the scopeof the appended claims. All publications and patent documents citedherein are hereby incorporated by reference in their entirety for allpurposes to the same extent as if each were so individually denoted.

1. A zinc finger protein that binds to a target site in a nucleic acid,the zinc finger protein comprising a plurality of zinc fingers, whereinone or more of the zinc fingers comprise the sequence shown in SEQ IDNO:17 or SEQ ID NO:19.
 2. The zinc finger protein of claim 1, whereinthe zinc finger protein comprises first, second and third zinc fingersand further wherein the first zinc finger comprises SEQ ID NO:17, thesecond zinc finger comprises SEQ ID NO:18 and the third zinc fingercomprises SEQ ID NO:19.
 3. A fusion protein comprising the zinc fingerprotein of claim 1 and a functional domain.
 4. An isolatedpolynucleotide encoding a zinc finger protein according to claim
 1. 5.An isolated polynucleotide encoding a zinc finger protein according toclaim
 2. 6. An expression vector comprising the isolated polynucleotideof claim
 4. 7. An expression vector comprising the isolatedpolynucleotide of claim
 5. 8. An isolated host cell comprising theisolated polynucleotide of claim
 4. 9. An isolated host cell comprisingthe isolated polynucleotide of claim 5.